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OBJECTIVE: IBD is characterised by dysbiosis, but it remains unclear to what extent dysbiosis develops in unaffected at-risk individuals. To address this, we investigated age-related patterns of faecal and serum markers of dysbiosis in high-risk multiplex IBD families (two or more affected first-degree relatives). DESIGN: Faecal and serum samples were collected from multiplex IBD and control families (95 IBD, 292 unaffected, 51 controls). Findings were validated in independent cohorts of 616 and 1173 subjects including patients with IBD, infants born to mothers with IBD and controls. 16S rRNA gene sequencing and global untargeted metabolomics profiling of faeces and serum were performed. RESULTS: Microbial and metabolomic parameters of dysbiosis progressively decreased from infancy until age 8. This microbial maturation process was slower in infants born to mothers with IBD. After age 15, dysbiosis steadily increased in unaffected relatives throughout adulthood. Dysbiosis was accompanied by marked shifts in the faecal metabolome and, to a lesser extent, the serum metabolome. Faecal and serum metabolomics dysbiosis indices were validated in an independent cohort. Dysbiosis was associated with elevated antimicrobial serologies but not with faecal calprotectin. Dysbiosis metrics differentiated IBD from non-IBD comparably to serologies, with a model combining calprotectin, faecal metabolomics dysbiosis index and serology score demonstrating highest accuracy. CONCLUSION: These findings support that dysbiosis exists as a pre-disease state detectable by faecal and serum biomarkers for IBD risk prediction. Given the expansion of disease-modifying agents and non-invasive imaging, the indices developed here may facilitate earlier diagnoses and improved management in at-risk individuals.
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The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis-specific SNPs were enriched in distal colon-related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.
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Colitis Ulcerosa , ARN Largo no Codificante , Humanos , Redes Reguladoras de Genes , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factor de Necrosis Tumoral alfa/genética , Colitis Ulcerosa/genética , InflamaciónRESUMEN
Studies have reported increased intestinal permeability in multiple sclerosis (MS) patients and its mouse model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms driving increased intestinal permeability that in turn exacerbate neuroinflammation during EAE remain unclear. Here we showed that vancomycin preserved the integrity of the intestinal barrier, while also suppressing gut trypsin activity, enhancing the relative abundance of specific Lactobacilli and ameliorating disease during EAE. Furthermore, Lactobacilli enriched in the gut of vancomycin-treated EAE mice at day 3 post immunization negatively correlated with gut trypsin activity and EAE severity. In untreated EAE mice, we observed increased intestinal permeability and increased intestinal protease activated receptor 2 (PAR2) expression at day 3 post immunization. Prior studies have shown that trypsin increases intestinal permeability by activating PAR2. Our results suggest that the interaction between intestinal PAR2 and trypsin may be a key modulator of intestinal permeability and disease severity during EAE.
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BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. METHODS: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). FINDINGS: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1ß, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. INTERPRETATION: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. FUNDING: None.
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Artritis Reumatoide , Microbioma Gastrointestinal , Ratones , Animales , Linfocitos T Reguladores , Magnesio/metabolismo , Magnesio/farmacología , Interleucina-10/genética , Interleucina-10/metabolismo , Citocinas/metabolismo , Artritis Reumatoide/metabolismo , Ratones Noqueados , Células Th17 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
Common variable immunodeficiency (CVID) is characterized by profound primary antibody defects and frequent infections, yet autoimmune/inflammatory complications of unclear origin occur in 50% of individuals and lead to increased mortality. Here, we show that circulating bacterial 16S rDNA belonging to gut commensals was significantly increased in CVID serum (P < 0.0001), especially in patients with inflammatory manifestations (P = 0.0007). Levels of serum bacterial DNA were associated with parameters of systemic immune activation, increased serum IFN-γ, and the lowest numbers of isotype-switched memory B cells. Bacterial DNA was bioactive in vitro and induced robust host IFN-γ responses, especially among patients with CVID with inflammatory manifestations. Patients with X-linked agammaglobulinemia (Bruton tyrosine kinase [BTK] deficiency) also had increased circulating bacterial 16S rDNA but did not exhibit prominent immune activation, suggesting that BTK may be a host modifier, dampening immune responses to microbial translocation. These data reveal a mechanism for chronic immune activation in CVID and potential therapeutic strategies to modify the clinical outcomes of this disease.
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Agammaglobulinemia/sangre , Inmunodeficiencia Variable Común/sangre , ADN Bacteriano/sangre , ADN Ribosómico/sangre , Microbioma Gastrointestinal/genética , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Inflamación/sangre , Adolescente , Adulto , Agammaglobulinemia/inmunología , Anciano , Anemia Hemolítica Autoinmune/sangre , Anemia Hemolítica Autoinmune/complicaciones , Anemia Hemolítica Autoinmune/inmunología , Linfocitos B/inmunología , Traslocación Bacteriana , Niño , Preescolar , Inmunodeficiencia Variable Común/complicaciones , Inmunodeficiencia Variable Común/inmunología , ADN Bacteriano/inmunología , ADN Ribosómico/inmunología , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Granuloma/sangre , Granuloma/complicaciones , Granuloma/inmunología , Humanos , Cambio de Clase de Inmunoglobulina , Memoria Inmunológica/inmunología , Inflamación/inmunología , Interferón gamma/sangre , Enfermedades Pulmonares Intersticiales/sangre , Enfermedades Pulmonares Intersticiales/complicaciones , Enfermedades Pulmonares Intersticiales/inmunología , Masculino , Persona de Mediana Edad , Poliendocrinopatías Autoinmunes/sangre , Poliendocrinopatías Autoinmunes/complicaciones , Poliendocrinopatías Autoinmunes/inmunología , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/complicaciones , Púrpura Trombocitopénica Idiopática/inmunología , Esplenomegalia/sangre , Esplenomegalia/complicaciones , Esplenomegalia/inmunología , Adulto JovenRESUMEN
Fecal microbiota transplantation (FMT) has been successfully applied to treat recurrent Clostridium difficile infection in humans, but a precise method to measure which bacterial strains stably engraft in recipients and evaluate their association with clinical outcomes is lacking. We assembled a collection of >1,000 different bacterial strains that were cultured from the fecal samples of 22 FMT donors and recipients. Using our strain collection combined with metagenomic sequencing data from the same samples, we developed a statistical approach named Strainer for the detection and tracking of bacterial strains from metagenomic sequencing data. We applied Strainer to evaluate a cohort of 13 FMT longitudinal clinical interventions and detected stable engraftment of 71% of donor microbiota strains in recipients up to 5 years post-FMT. We found that 80% of recipient gut bacterial strains pre-FMT were eliminated by FMT and that post-FMT the strains present persisted up to 5 years later, together with environmentally acquired strains. Quantification of donor bacterial strain engraftment in recipients independently explained (precision 100%, recall 95%) the clinical outcomes (relapse or success) after initial and repeat FMT. We report a compendium of bacterial species and strains that consistently engraft in recipients over time that could be used in defined live biotherapeutic products as an alternative to FMT. Our analytical framework and Strainer can be applied to systematically evaluate either FMT or defined live bacterial therapeutic studies by quantification of strain engraftment in recipients.
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Bacterias/aislamiento & purificación , Trasplante de Microbiota Fecal , Algoritmos , Bacterias/clasificación , Bacterias/genética , Benchmarking , Clostridioides difficile/fisiología , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/terapia , Trasplante de Microbiota Fecal/métodos , Heces/microbiología , Microbioma Gastrointestinal , Humanos , Estudios Longitudinales , Metagenoma/genética , Recurrencia , Donantes de Tejidos , Resultado del TratamientoRESUMEN
Gastrointestinal symptoms are common in COVID-19 patients but the nature of the gut immune response to SARS-CoV-2 remains poorly characterized, partly due to the difficulty of obtaining biopsy specimens from infected individuals. In lieu of tissue samples, we measured cytokines, inflammatory markers, viral RNA, microbiome composition, and antibody responses in stool samples from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.
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COVID-19 , Heces , Microbioma Gastrointestinal , Nasofaringe/virología , ARN Viral/aislamiento & purificación , Anciano , Biomarcadores/metabolismo , COVID-19/epidemiología , COVID-19/inmunología , Estudios de Cohortes , Citocinas/metabolismo , Heces/virología , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Masculino , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , SARS-CoV-2/aislamiento & purificaciónRESUMEN
The presence of an immunosuppressive tumor microenvironment is a major obstacle in the success of cancer immunotherapies. Because extracellular matrix components can shape the microenvironment, we investigated the role of matrix metalloproteinase 2 (MMP2) in melanoma tumorigenesis. We found that MMP2 signals proinflammatory pathways on antigen presenting cells, and this requires both TLR2 and TLR4. B16 melanoma cells that express MMP2 at baseline have slower kinetics in Tlr2-/- Tlr4-/- mice, implicating MMP2 in promoting tumor growth. Indeed, Mmp2 overexpression in B16 cells potentiated rapid tumor growth, which was accompanied by reduced intratumoral cytolytic cells and increased M2 macrophages. In contrast, knockdown of Mmp2 slowed tumor growth and enhanced T cell proliferation and NK cell recruitment. Finally, we found that these effects of MMP2 are mediated through dysfunctional DC-T cell cross-talk as they are lost in Batf3-/- and Rag2-/- mice. These findings provide insights into the detrimental role of endogenous alarmins like MMP2 in modulating immune responses in the tumor microenvironment.
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Metaloproteinasa 2 de la Matriz/inmunología , Receptores Toll-Like/inmunología , Microambiente Tumoral/inmunología , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Both genetic predisposition and environmental factors appear to play a role in inflammatory bowel disease (IBD) development. Genetic studies in humans have linked the interleukin (IL)-23 signaling pathway with IBD, but the environmental factors contributing to disease have remained elusive. Here, we show that the azo dyes Red 40 and Yellow 6, the most abundant food colorants in the world, can trigger an IBD-like colitis in mice conditionally expressing IL-23, or in two additional animal models in which IL-23 expression was augmented. Increased IL-23 expression led to generation of activated CD4+ T cells that expressed interferon-γ and transferred disease to mice exposed to Red 40. Colitis induction was dependent on the commensal microbiota promoting the azo reduction of Red 40 and generation of a metabolite, 1-amino-2-naphthol-6-sulfonate sodium salt. Together these findings suggest that specific food colorants represent novel risk factors for development of colitis in mice with increased IL-23 signaling.
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Bacterias/metabolismo , Colitis , Colorantes de Alimentos/metabolismo , Interleucina-23/genética , Mucosa Intestinal/microbiología , Animales , Colitis/genética , Colitis/metabolismo , Colitis/microbiología , Modelos Animales de Enfermedad , Colorantes de Alimentos/efectos adversos , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Interferón gamma/genética , Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , SimbiosisRESUMEN
PURPOSE: Immunoprofiling to identify biomarkers and integration with clinical trial outcomes are critical to improving immunotherapy approaches for patients with cancer. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assay performance further limits comparison and interpretation of data across studies and laboratories. EXPERIMENTAL DESIGN: To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer MoonshotSM Initiative of the National Cancer Institute (NCI) and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the NIH. RESULTS: The CIMAC-CIDC Network is composed of four academic centers with multidisciplinary expertise in cancer immunotherapy that perform validated and harmonized assays for immunoprofiling and conduct correlative analyses. A data coordinating center (CIDC) provides the computational expertise and informatics platforms for the storage, integration, and analysis of biomarker and clinical data. CONCLUSIONS: This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure, validation and harmonization of core immunoprofiling assays, platforms for data ingestion and integration, and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole-exome sequencing, RNA sequencing, cytometry by time of flight, and IHC/immunofluorescence.
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Biomarcadores de Tumor/inmunología , Inmunoterapia , Monitorización Inmunológica , Neoplasias/inmunología , Neoplasias/terapia , HumanosRESUMEN
We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.
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The building evidence for the contribution of microbiota to human disease has spurred an effort to develop therapies that target the gut microbiota. This is particularly evident in inflammatory bowel diseases (IBDs), where clinical trials of fecal microbiota transplantation have shown some efficacy. To aid the development of novel microbiota-targeted therapies and to better understand the biology underpinning such treatments, we have used gnotobiotic mice to model microbiota manipulations in the context of microbiotas from humans with inflammatory bowel disease. Mice colonized with IBD donor-derived microbiotas exhibit a stereotypical set of phenotypes, characterized by abundant mucosal Th17 cells, a deficit in the tolerogenic RORγt+ regulatory T (Treg) cell subset, and susceptibility to disease in colitis models. Transplanting healthy donor-derived microbiotas into mice colonized with human IBD microbiotas led to induction of RORγt+ Treg cells, which was associated with an increase in the density of the microbiotas following transplant. Microbiota transplant reduced gut Th17 cells in mice colonized with a microbiota from a donor with Crohn's disease. By culturing strains from this microbiota and screening them in vivo, we identified a specific strain that potently induces Th17 cells. Microbiota transplants reduced the relative abundance of this strain in the gut microbiota, which was correlated with a reduction in Th17 cells and protection from colitis.
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Trasplante de Microbiota Fecal , Enfermedades Inflamatorias del Intestino/microbiología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Colitis/prevención & control , Colon/microbiología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/microbiología , Citocinas/inmunología , Modelos Animales de Enfermedad , Heces/microbiología , Femenino , Microbioma Gastrointestinal/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/microbiología , Células Th17/microbiologíaRESUMEN
BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.
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Pólipos del Colon/inmunología , Receptores ErbB/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/patología , Metaloproteinasa 3 de la Matriz/metabolismo , Animales , Apoptosis/inmunología , Ciego/citología , Ciego/inmunología , Ciego/patología , Toxina Diftérica/administración & dosificación , Toxina Diftérica/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/patología , Receptores ErbB/antagonistas & inhibidores , Fibroblastos/inmunología , Fibroblastos/metabolismo , Gefitinib/farmacología , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Interleucina-1beta/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Metaloproteinasa 3 de la Matriz/inmunología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Transgénicos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Increased expression of Interleukin (IL)-33 has been detected in intestinal samples of patients with ulcerative colitis, a condition associated with increased risk for colon cancer, but its role in the development of colorectal cancer has yet to be fully examined. Here, we investigated the role of epithelial expressed IL-33 during development of intestinal tumors. IL-33 expression was detected in epithelial cells in colorectal cancer specimens and in the Apc Min/+ mice. To better understand the role of epithelial-derived IL-33 in the intestinal tumorigenesis, we generated transgenic mice expressing IL-33 in intestinal epithelial cells (V33 mice). V33 Apc Min/+ mice, resulting from the cross of V33 with Apc Min/+ mice, had increased intestinal tumor burden compared with littermate Apc Min/+ mice. Consistently, Apc Min/+ mice deficient for IL-33 receptor (ST2), had reduced polyp burden. Mechanistically, overexpression of IL-33 promoted expansion of ST2+ regulatory T cells, increased Th2 cytokine milieu, and induced alternatively activated macrophages in the gut. IL-33 promoted marked changes in the expression of antimicrobial peptides, and antibiotic treatment of V33 Apc Min/+ mice abrogated the tumor promoting-effects of IL-33 in the colon. In conclusion, elevated IL-33 signaling increases tumor development in the Apc Min/+ mice.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Transformación Celular Neoplásica/inmunología , Células Epiteliales/metabolismo , Interleucina-33/metabolismo , Neoplasias Intestinales/inmunología , Animales , Transformación Celular Neoplásica/genética , Células Cultivadas , Citocinas/metabolismo , Células Epiteliales/citología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Neoplasias Intestinales/genética , Activación de Macrófagos , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Transducción de Señal , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Regulación hacia ArribaRESUMEN
Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4+ T-cell activation. A common 'suppression of inflammation' signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4+ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.
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Apoptosis , Células Epiteliales/citología , Células Epiteliales/inmunología , Homeostasis , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Fagocitos/citología , Fagocitos/inmunología , Aminoácidos/metabolismo , Animales , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Movimiento Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Cadenas alfa de Integrinas/metabolismo , Metabolismo de los Lípidos , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Fagocitos/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Transcripción GenéticaRESUMEN
Interleukin-23 (IL-23) responsive group 3 innate lymphoid cells (ILC3s) have been implicated in immune homeostasis and pathogenesis in the adult, but little is known about their roles in the newborn. Here we show that IL-23 promotes conversion of embryonic intestinal Lin(-)IL-23R(+)Thy1(+) cells into IL-22-producing Thy1(+)Sca-1(hi) ILC3s in vitro. Gut-specific expression of IL-23 also activated and expanded Thy1(+)Sca-1(hi) ILC3s, which produced IL-22, IL-17, interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) and were distinct from canonical CD4(+) lymphoid tissue inducer (LTi) cells. These ILC3s accumulated under the epithelium in intercellular adhesion molecule (ICAM)-1-positive cell aggregates together with neutrophils that disrupted the epithelium, leading to the formation of discrete intestinal erosions, bleeding, and neonatal death. Genetic and antibody depletion of ILC3s rescued the mice from neonatal death. Antibiotic treatment of pregnant mothers and offspring prolonged survival of IL-23 transgenic mice, suggesting a role for the commensal flora on ILC3-induced pathogenesis. Our results reveal a novel role for the IL-23-ILC3s axis in the pathogenesis of neonatal intestinal inflammation.
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Inmunidad Innata , Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Animales , Antígenos de Superficie/metabolismo , Citocinas/metabolismo , Expresión Génica , Humanos , Inmunidad Innata/genética , Inmunofenotipificación , Interleucina-23/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Intestinos/microbiología , Intestinos/patología , Ratones , Ratones Transgénicos , Infiltración Neutrófila , Neutrófilos/inmunología , Neutrófilos/metabolismo , Permeabilidad , FenotipoRESUMEN
The preferential localization of some neoplasms, such as serrated polyps (SPs), in specific areas of the intestine suggests that nongenetic factors may be important for their development. To test this hypothesis, we took advantage of transgenic mice that expressed HB-EGF throughout the intestine but developed SPs only in the cecum. Here we show that a host-specific microbiome was associated with SPs and that alterations of the microbiota induced by antibiotic treatment or by embryo transfer rederivation markedly inhibited the formation of SPs in the cecum. Mechanistically, development of SPs was associated with a local decrease in epithelial barrier function, bacterial invasion, production of antimicrobials, and increased expression of several inflammatory factors such as IL-17, Cxcl2, Tnf-α, and IL-1. Increased numbers of neutrophils were found within the SPs, and their depletion significantly reduced polyp growth. Together these results indicate that nongenetic factors contribute to the development of SPs and suggest that the development of these intestinal neoplasms in the cecum is driven by the interplay between genetic changes in the host, an inflammatory response, and a host-specific microbiota.
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Ciego/patología , Epitelio/fisiología , Regulación Neoplásica de la Expresión Génica/inmunología , Pólipos Intestinales/patología , Microbiota/fisiología , Modelos Moleculares , Animales , Antibacterianos/farmacología , Citocinas/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pólipos Intestinales/metabolismo , Ratones , Ratones Transgénicos , Conformación Proteica , Especificidad de la EspecieRESUMEN
Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.
Asunto(s)
Citomegalovirus/fisiología , Receptores de Quimiocina/metabolismo , Proteínas Virales/metabolismo , Fusión Artificial Génica , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/aislamiento & purificación , Citoplasma/química , Células Endoteliales/virología , Células Epiteliales/virología , Fibroblastos/virología , Eliminación de Gen , Genes Reporteros , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Receptores de Quimiocina/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Coloración y Etiquetado , Proteínas Virales/genéticaRESUMEN
BACKGROUND & AIMS: Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. METHODS: Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. RESULTS: EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. CONCLUSIONS: Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas.